Project Summary: This proposal aims to clarify the catalytic role of an active site adenosine in the Varkud Satellite (VS) ribozyme. RNA-based enzymes (ribozymes) perform critical functions in a wide variety of cellular processes, including protein synthesis, tRNA maturation and mRNA splicing. An improved understanding of ribozyme catalysis will aid in the design of ribozyme inhibitors, and should improve efforts to engineer new ribozymes as targeted gene splicing elements, with applications as anti-cancer or anti-viral agents. Several proposed ribozyme mechanisms utilize nucleobase nitrogens as sites of proton transfer, analogous to the role of histidine in protein enzymes. These mechanisms require large active site perturbations to nucleobase pK(a) values. The broad goal of this proposal is to design a series of adenosine analogues that can be used to directly test the importance of adenosine protonation in ribozyme catalysis. The specific aims of the proposal are directed toward exploring the mechanism of the reversible cleavage/ligation reaction of the VS ribozyme, where several lines of evidence suggest adenosine 756 is protonated at some stage of the catalytic cycle. The proposed analogues make fluorine for hydrogen substitutions in adenosine or 7-deaza-adenosine. The introduction of fluorine provides both a means of perturbing adenenosine's pK(a) and a unique spectroscopic observable with which to monitor analogue protonation state. The proposed methods outline a strategy to incorporate a series of fluoroadenosines site-specifically at A756 of a previously described semi-synthetic construct. The four analogues chosen will produce a range of N1 pK(a) values at A756, from well below to slightly above that of adenosine, with minimal structural perturbation. The rates of both the cleavage and ligation reactions will be measured as a function of pH with each of these analogues incorporated at A756 (Aim 1). Reactant stabilized versions of the same constructs will then be used to determine the protonation state of each analogue as a function of pH by (19)F NMR (Aim 2). By combining pH dependent rates for each analogue with an independent measure of analogue protonation the proposed research will provide a direct test of the importance of A756 protonation in the mechanism of VS ribozyme cleavage/ligation. Relevance: This proposal investigates the mechanism by which a particular ribozyme (RNA based enzyme) performs its function. Ribozymes perform a variety of critical functions, both in human biology and in the biology of human pathogens. An increased understanding of these mechanisms will aid the development of drugs to combat human pathogens and in the engineering of new ribozymes with therapeutic applications.